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Rubisco of a Nucleoside Pathway Known from Archaea Is Found in Diverse Uncultivated Phyla in Bacteria

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Rubisco of a Nucleoside Pathway Known from Archaea Is Found in Diverse Uncultivated Phyla in Bacteria Lawrence Berkeley National Laboratory Recent Work Title RubisCO of a nucleoside pathway known from Archaea is found in diverse uncultivated phyla in bacteria. Permalink https://escholarship.org/uc/item/4dk6g95j Journal The ISME journal, 10(11) ISSN 1751-7362 Authors Wrighton, Kelly C Castelle, Cindy J Varaljay, Vanessa A et al. Publication Date 2016-11-01 DOI 10.1038/ismej.2016.53 Peer reviewed eScholarship.org Powered by the California Digital Library University of California The ISME Journal (2016) 10, 2702–2714 © 2016 International Society for Microbial Ecology All rights reserved 1751-7362/16 OPEN www.nature.com/ismej ORIGINAL ARTICLE RubisCO of a nucleoside pathway known from Archaea is found in diverse uncultivated phyla in bacteria Kelly C Wrighton1,7, Cindy J Castelle2,7, Vanessa A Varaljay1, Sriram Satagopan1, Christopher T Brown3, Michael J Wilkins1,4, Brian C Thomas2, Itai Sharon2, Kenneth H Williams5, F Robert Tabita1 and Jillian F Banfield2,6 1Department of Microbiology, The Ohio State University, Columbus, OH, USA; 2Department of Earth and Planetary Science, UC Berkeley, Berkeley, CA, USA; 3Department of Plant and Microbial Biology, UC Berkeley, Berkeley, CA, USA; 4School of Earth Sciences, The Ohio State University, Columbus, OH, USA; 5Earth Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA and 6Department of Environmental Science, Policy, and Management, University of California, Berkeley, CA, USA Metagenomic studies recently uncovered form II/III RubisCO genes, originally thought to only occur in archaea, from uncultivated bacteria of the candidate phyla radiation (CPR). There are no isolated CPR bacteria and these organisms are predicted to have limited metabolic capacities. Here we expand the known diversity of RubisCO from CPR lineages. We report a form of RubisCO, distantly similar to the archaeal form III RubisCO, in some CPR bacteria from the Parcubacteria (OD1), WS6 and Microgenomates (OP11) phyla. In addition, we significantly expand the Peregrinibacteria (PER) II/III RubisCO diversity and report the first II/III RubisCO sequences from the Microgenomates and WS6 phyla. To provide a metabolic context for these RubisCOs, we reconstructed near-complete (493%) PER genomes and the first closed genome for a WS6 bacterium, for which we propose the phylum name Dojkabacteria. Genomic and bioinformatic analyses suggest that the CPR RubisCOs function in a nucleoside pathway similar to that proposed in Archaea. Detection of form II/III RubisCO and nucleoside metabolism gene transcripts from a PER supports the operation of this pathway in situ. We demonstrate that the PER form II/III RubisCO is catalytically active, fixing CO2 to physiologically complement phototrophic growth in a bacterial photoautotrophic RubisCO deletion strain. We propose that the identification of these RubisCOs across a radiation of obligately fermentative, small- celled organisms hints at a widespread, simple metabolic platform in which ribose may be a prominent currency. The ISME Journal (2016) 10, 2702–2714; doi:10.1038/ismej.2016.53; published online 3 May 2016 Introduction (CPR), a monophyletic group of at least 35 phyla that accounts for 415% of all bacterial diversity (Brown The vast majority of the organisms in the environment et al., 2015). Metagenomic sampling of almost 800 have not been cultivated, obscuring our knowledge of CPR bacteria suggested that members of this radia- their physiology. Recent metagenomic investigations tion consistently have small genomes with many haverevealedthepresenceofalargediversityof metabolic limitations, including lack of an electron uncultivated organisms in marine and terrestrial transport chain, no more than a partial tricarboxylic subsurface environments (Castelle et al., 2013, 2015; acid cycle and mostly incomplete nucleotide and Lloyd et al.,2013;Bakeret al., 2015). For example, in a amino-acid biosynthesis pathways. Using metabolic metagenomic study of a shallow alluvial aquifer, we predictions from complete and near-complete gen- determined that many of the uncultivated bacteria omes (Wrighton et al., 2012), we inferred that were associated with the candidate phyla radiation members of the CPR are fermenters whose primary biogeochemical impact is primarily on subsurface Correspondence: JF Banfield, Department of Earth and Planetary organic carbon and hydrogen cycling (Wrighton Sciences, and Department of Environmental Science, Policy, and et al., 2014). However, many of the genes in these Man, University of California at Berkeley, 336 Hilgard Hall, genomes remain poorly annotated and the metabolic Berkeley, CA 94720, USA. platform of CPR bacteria remains uncertain. Further, E-mail: [email protected] 7These authors contributed equally to this work. no transcription and enzyme activities have been Received 25 August 2015; revised 29 February 2016; accepted validated, hindering knowledge of the actual reac- 4 March 2016; published online 3 May 2016 tions catalyzed by these organisms. RubicCO in uncultivated bacteria of the CPR KC Wrighton et al 2703 The incorporation of CO2 into organic carbon is a experiments were performed by adding approxi- critical step in the carbon cycle. Ribulose-1,5- mately 15 mM acetate to the groundwater over the bisphophate carboxylase-oxygenase (RubisCO) is course of 72 days to maintain an average in situ the most abundant enzyme on earth and is integral concentration of ~ 3 mM acetate during peak stimula- to the fixation of carbon dioxide. Currently four tion. Six microbial community samples (A–F) were forms of RubisCO can fix atmospheric carbon collected over 93 days, including 3 days prior to dioxide (I, II, II/III and III) (Tabita et al., 2007; acetate stimulation and after acetate addition ceased 2008). Forms I and II are used by plants, algae and (amendment ceased on day 72). Ferrous iron and some chemoautotrophic and phototrophic bacteria sulfide concentrations were analyzed immediately for CO2 fixation during primary production via the after sampling using the HACH phenanthroline assay Calvin Benson Bassham (CBB) cycle. Forms III and and methylene blue sulfide reagent kit, respectively II/III, found primarily in archaea, enable light- (HACH, Loveland, CO, USA). Acetate and sulfate independent CO2 incorporation into sugars derived concentrations were determined using a Dionex from nucleotides like adenosine monophosphate ICS-2100 ion chromatograph (Sunnyvale, CA, USA) (AMP) (Sato et al., 2007; Tabita et al., 2007). Form equipped with an AS-18 guard and analytical column II/III RubisCOs, which were primarily known in (Williams et al., 2011). Microbial cells from pumped methanogens, have greater overall similarity to groundwater that passed through a 1.2-μm prefilter bacterial Form II RubisCO but have specific residues, (Pall, Port Washington, NY, USA) were retained on 0.2- structural and catalytic features that more closely and 0.1-μm filters (Pall). Filters were flash-frozen in resemble archaeal form III (Alonso et al., 2009). liquid nitrogen immediately upon collection. Previously, we reported the first evidence of form II/III RubisCO genes in partial genomes from the Peregrinibacteria (PER) phylum in the CPR radiation Nucleic acid extraction and sequencing (Wrighton et al., 2012). Additionally, three bacterial Approximately a 1.5-g sample from each frozen filter form II/III RubisCOs were recovered from genomes of was used for genomic DNA extraction. DNA was members of the SR1 phylum that also are inferred to extracted using the PowerSoil DNA Isolation Kit be fermentative (Wrighton et al., 2012; Campbell (MoBio Laboratories, Inc., Carlsbad, CA, USA). et al., 2013; Kantor et al., 2013). The presence of Illumina HiSeq 2000 2 × 150 paired-end sequencing these annotated RubisCO genes in small genomes was conducted by the Joint Genome Institute on that house few ancillary genes suggests that RubisCO extracted DNA. Sequencing amounts for samples A– may have an important role in the metabolism of F from 0.1- and 0.2-μm filters ranged from 9.5 to some of these bacteria. 40.7 Gbp, as described in Castelle et al. (2015). RNA To more comprehensively explore the diversity was extracted using Invitrogen TRIzol Reagent and potential role of CPR RubisCOs, we reproduced (Carlsbad, CA, USA) followed by genomic DNA the prior experiment from which we first identified removal and cleaning using Qiagen RNase-Free bacterial form II/III RubisCO sequences (Wrighton DNase Set Kit (Valencia, CA, USA) and Qiagen Mini et al., 2012). Size filtration was used to increase the RNeasy Kit (Valencia, CA, USA). An Agilent 2100 relative abundance of the small-celled CPR organ- Bioanalyzer (Santa Clara, CA, USA) was used to isms in samples used for sequencing (Luef et al., assess the integrity of the RNA samples. Only RNA 2015). Here we investigate CPR genomes that encode samples having RNA Integrity Number between 8 RubisCO genes, report a new RubisCO sequence type and 10 were used. The Applied Biosystems SOLiD diverged from Archaeal form III and propose a Total RNA-Seq Kit (Invitrogen/LifeTechnologies, broader metabolic context in which these genes Carlsbad, CA, USA) generated the cDNA template occur. By detecting in situ transcripts and through library. The SOLiD EZ Bead system was used to directly cloning one CPR form II/III RubisCO perform emulsion clonal bead amplification to sequence into an expression host, we suggest the generate bead templates for SOLiD platform sequen- potential functionality of this bacterial enzyme. This cing. Samples were sequenced
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